Document QAS/11.

452 FINAL
July 2012

3.4 TEST FOR BACTERIAL ENDOTOXINS

Final text for revision of The International Pharmacopoeia

This monograph was adopted at the Forty-sixth WHO Expert Committee on Specifications for
Pharmaceutical Preparations in October 2011 for addition to the 4th Edition of the International
Pharmacopoeia

The text, reproduced with the permission of the Japanese Pharmacopoeia with appropriate
editorial modifications, is one that has undergone pharmacopoeial harmonization by the
Pharmacopoeial Discussion Group (PDG) of the European Pharmacopoeia (Ph.Eur), Japanese
Pharmacopoeia (JP) and United States Pharmacopeia (USP).

3.4 – TEST FOR BACTERIAL ENDOTOXINS

The bacterial endotoxins test (BET) is a test to detect or quantify endotoxins from Gram-
negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or
Tachypleus tridentatus). There are three methods for this test:

• Method A. The gel-clot technique, which is based on gel formation;

• Method B. The turbidimetric technique, based on the development of turbidity after
cleavage of an endogenous substrate;
• Method C. The chromogenic technique, based on the development of color after
cleavage of a synthetic peptide-chromogen complex.

Unless otherwise indicated in the individual monograph proceed by Method A.

The test is carried out in a manner that avoids endotoxin contamination.

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APPARATUS

Depyrogenate all glassware and other heat stable materials in a hot air oven using a validated
process. A commonly used minimum time and temperature is 30 minutes at 250°C. If
employing plastic apparatus such as microplates and pipet tips for automatic pipetters, use
apparatus shown to be free of detectable endotoxin and which does not interfere in the test.

Note: In this chapter the term "tube" includes any other receptacle such as a micro-titre well.

REAGENTS AND TEST SOLUTIONS

 Amoebocyte lysate
A lyophilized product obtained from the lysate of amebocytes (white blood cells) from
the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).
Note: Amebocyte lysate reacts to some β-glucans in addition to endotoxins.
Amebocyte lysate preparations which do not react to glucans are available: they are
prepared by removing the G factor reacting to glucans from amebocyte lysate or by
inhibiting the G factor reacting system of amebocyte lysate and may be used for the
endotoxin testing in the presence of glucans.

 Lysate TS
Dissolve amebocyte lysate in water BET or in a buffer recommended by the lysate
manufacturer, by gentle stirring. Store the reconstituted lysate, refrigerated or frozen,
according to the specifications of the manufacturer.

 Water BET

Water for injections or water produced by other procedures that shows no reaction with
the lysate employed, at the detection limit of the reagent.
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Preparation of Standard Endotoxin Stock Solution

A Standard Endotoxin Stock Solution is prepared from an endotoxin reference standard that
has been calibrated against the International Standard for endotoxins. Follow the specifications
in the package leaflet and on the label for preparation and storage of the Standard Endotoxin
Stock Solution.

Endotoxin is expressed in International Units (IU) of endotoxin.

Note: One International Unit (IU) of endotoxin is equal to one Endotoxin Unit (EU).

Preparation of Standard Endotoxin Solution

After mixing the Standard Endotoxin Stock Solution vigorously, prepare appropriate serial
dilutions of Standard Endotoxin Solution, using water BET.

Use dilutions as soon as possible to avoid loss of activity by adsorption.

Preparation of sample solutions

Prepare sample solutions by dissolving or diluting drugs using water BET. Some substances or
preparations may be more appropriately dissolved or diluted in other aqueous solutions. If
necessary, adjust the pH of the solution to be examined (or dilution thereof) so that the pH of
the mixture of the lysate TS and sample solution falls within the pH range specified by the
lysate manufacturer, usually 6.0 to 8.0. The pH may be adjusted by the use of acid, base, or
suitable buffer as recommended by the lysate manufacturer. Acids and bases may be prepared
from concentrates or solids with water BET in containers free of detectable endotoxin. Buffers
must be validated to be free of detectable endotoxin and interfering factors.

DETERMINATION OF MAXIMUM VALID DILUTION

The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample at which
the endotoxin limit can be determined.

Determine the MVD from the following equation:

Endotoxin Limit × Concentration of sample solution

MVD =
λ
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Endotoxin Limit:

The endotoxin limit for parenteral preparations, defined on the basis of dose, equals K/M,
where K is a threshold pyrogenic dose of endotoxin per kg of body weight, and M is equal to
the maximum recommended bolus dose of product per kg of body weight. When the product is
to be injected at frequent intervals or infused continuously, M is the maximum total dose
administered in a single hour period.

Note: The endotoxin limit depends on the product and its route of administration and is stated
in the individual monograph. Suggested values for K are:

- intravenous route: K = 5 IU endotoxin per kg body weight;

- intravenous route for radiopharmaceuticals: K = 2.5 IU endotoxin per kg body weight;
- intrathecal route: K = 0.2 IU endotoxin per kg body weight.

For other routes of administration, the acceptance criterion for bacterial endotoxins is generally
determined on the basis of results obtained during development of the preparation.
The endotoxin limit for parenteral preparations is specified in units such as IU/ml, IU/mg,
IU/Unit of biological activity, etc., in the individual monograph.

Concentration of sample solution:

- mg/ml in the case of endotoxin limit specified by weight (IU/mg)
- units/ml in the case of endotoxin limit specified by unit of biological activity (IU/Unit)
- ml/ml when the endotoxin limit is specified by volume (IU/ml)

λ: the labeled lysate sensitivity in the gel-clot technique (IU/ml) or the lowest concentration
used in the standard curve for the turbidimetric or chromogenic techniques.

METHOD A: GEL-CLOT TECHNIQUE

The gel-clot technique is for detecting or quantifying endotoxins based on clotting of the lysate
TS in the presence of endotoxin. The minimum concentration of endotoxin required to cause
the lysate to clot under standard conditions is the labeled sensitivity of the lysate TS. To
ensure both the precision and validity of the test, perform the tests for confirming the labeled
lysate sensitivity and for interfering factors as described below under Preparatory testing.

Preparatory testing

Test for confirmation of labeled lysate sensitivity

Confirm in four replicates the labeled sensitivity, λ, expressed in IU/ml of the lysate prior to
use in the test. The test for confirmation of the lysate sensitivity is to be carried out when a
new lot of lysate is used or when there is any change in the test conditions which may affect
the outcome of the test.
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Prepare standard solutions having at least four concentrations equivalent to 2λ , λ, 0.5λ and
0.25λ by diluting the Standard Endotoxin Stock Solution with water BET.

Mix a volume of the lysate TS with an equal volume of one of the standard solutions (such as
0.1 ml aliquots) in each tube. When single test vials or ampoules, containing lyophilized lysate
are employed, add solutions of standards directly to the vial or ampoule. Incubate the reaction
mixture for a constant period according to directions of the lysate manufacturer (usually at
37±1°C for 60 ± 2 minutes), avoiding vibration. Test the integrity of the gel for tests carried
out in tubes, take each tube in turn directly from the incubator and invert it through
approximately 180 degrees in one smooth motion. If a firm gel has formed that remains in
place upon inversion, record the result as positive. A result is negative if an intact gel is not
formed.

The test is considered valid when the lowest concentration of the standard solutions shows a
negative result in all replicate tests.

The endpoint is the lowest concentration in the series of decreasing concentrations of standard
endotoxin that clots the lysate. Determine the geometric mean endpoint concentration by
calculating the mean of the logarithms of the endpoint concentrations of the four dilution
series, take the antilogarithm of this value, as indicated in the following formula:

∑e
Geometric Mean Endpoint Concentration = antilog
f

∑ e = the sum of the log endpoint concentrations of the dilution series used
f = the number of replicate test tubes

The geometric mean endpoint concentration is the measured sensitivity of the lysate (IU/ml). If
this is not less than 0.5λ and not more than 2λ, the labeled sensitivity is confirmed and is used
in tests performed with this lysate.

Test for interfering factors

Usually prepare the solutions (A-D) in Table 1, and perform the inhibition/enhancement test on
the sample solutions at a dilution less than the MVD not containing any detectable endotoxins,
operating as described above under Test for confirmation of labeled lysate sensitivity.
The geometric mean endpoint concentrations of B and C solutions are determined by using the
formula described above under Test for confirmation of labeled lysate sensitivity.

The test for interfering factors must be repeated when any condition changes which is likely to
influence the result of the test.
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Table 1
Solution Endotoxin Diluent Dilution Endotoxin Number of
concentration/Solution to concentration replicates
which endotoxin is added factor

A None / Sample solution - - - 4

1 2λ 4

Sample 2 1λ 4
B 2λ / Sample solution
solution
4 0.5λ 4

8 0.25λ 4

1 2λ 2

Water 2 1λ 2
C 2 λ / Water BET
BET 4 2
0.5λ
8 0.25λ 2

D None / Water BET - - - 2

Note:
- Solution A : a sample solution of the preparation under test that is free of detectable
endotoxins
- Solution B : test for interference
- Solution C : control for labeled lysate sensitivity
- Solution D : negative control of water BET.
The test is considered valid when all replicates of solutions A and D show no reaction and the
result of solution C confirms the labeled sensitivity.

If the sensitivity of the lysate determined in the presence of solution B is not less than 0.5 and
not greater than 2λ, the sample solution does not contain factors which interfere under the
experimental conditions used. Otherwise the sample solution to be examined interferes with
the test.

If the preparation under test does not comply with the test at a dilution less than the MVD,
repeat the test using a greater dilution, not exceeding the MVD. The use of a more sensitive
lysate permits a greater dilution of the preparation to be examined and this may contribute to
the elimination of interference.

Interference may be overcome by suitable treatment, such as filtration, neutralization, dialysis

or heat treatment. To establish that the treatment chosen effectively eliminates interference
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without loss of endotoxins, perform the assay described above using the preparation to be
examined to which standard endotoxin has been added and which has then been submitted to
the chosen treatment.

Limit test

Procedure
Prepare the solutions A, B, C and D according to Table 2, and perform the test on these
solutions following the procedure in Test for confirmation of labeled lysate sensitivity under
Preparatory testing.

Table 2
Endotoxin concentration / Solution to which Number of
Solution
endotoxin is added replicates

A None / Diluted sample solution 2

B 2λ / Diluted sample solution 2

C 2λ / Water BET 2

D None / Water BET 2

Note: Prepare the solution A and the positive product control solution B using a dilution not
greater than the MVD and treatments as for the Test for interfering factors under
Preparatory testing. The positive control solutions B and C contain the standard
endotoxin preparation at a concentration corresponding to twice the labeled lysate
sensitivity. The negative control solution D consists of water BET.

Interpretation
The test is considered valid when both replicates of solution B and C are positive and those of
solution D are negative.

When a negative result is found for both replicates of solution A, the preparation under test
complies with the test.

When a positive result is found for both replicates of solution A, the preparation under test
does not comply with the test.

When a positive result is found for one replicate of solution A and a negative result is found for
the other, repeat the test. In the repeat test, the preparation under test complies with the test if a
negative result is found for both replicates of solution A. The preparation does not comply
with the test if a positive result is found for one or both replicates of solution A.

However, if the preparation does not comply with the test at a dilution less than the MVD, the
test may be repeated using a greater dilution, not exceeding the MVD.
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Quantitative Test

Procedure
The test quantifies bacterial endotoxins in sample solutions by titration to an endpoint.
Prepare the solutions A, B, C and D following Table 3, and test these solutions according to the
procedure in Test for confirmation of labeled lysate sensitivity under Preparatory testing.

Table 3
Solution Endotoxin Diluent Dilution Endotoxin Number of
concentration/Solution to factor concentration replicates
which endotoxin is added

1 - 2

Water 2 - 2
A None / Sample solution
BET 4 - 2

8 - 2

B 2λ / Sample solution 1 2λ 2

1 2λ 2

Water 2 1λ 2
C 2λ / Water BET
BET 4 0.5λ 2

8 0.25λ 2

D None / Water BET - - - 2

Note:
- Solution A: Sample solution under test at the dilution, not to exceed the MVD, with
which the test for interfering factors was completed. Subsequent dilution of the sample
solution must not exceed the MVD. Use water BET to make a dilution series of four
tubes containing the sample solution under test at concentrations of 1, 1/2, 1/4 and 1/8
relative to the concentration used in the test for interfering factors. Other dilutions up to
the MVD may be used as appropriate.
- Solution B: Solution A containing standard endotoxin at a concentration of 2 (positive
product control)
- Solution C: A dilution series of four tubes of water BET containing the standard
endotoxin at a concentration of 2λ, λ, 0.5λ and 0.25λ, respectively
- Solution D: Water BET (negative control)
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Calculation and interpretation

The test is considered valid when the following three conditions are met.
1: Both replicates of the negative control solution D are negative.
2: Both replicates of the positive product control solution B are positive.
3: The geometric mean endpoint concentration of the solution C is in the range of 0.5  to
2 .

To determine the endotoxin concentration of solution A, calculate the endpoint concentration

for each replicate by multiplying each endpoint dilution factor by λ.
The endotoxin concentration in the sample solution is the endpoint concentration of the
replicates. If the test is conducted with a diluted sample solution, calculate the concentration of
endotoxin in the original sample solution by multiplying by the dilution factor.

If none of the dilutions of sample solution is positive in a valid assay, report the endotoxin
concentration as less than λ (if diluted sample was tested, report as less than the smallest
dilution factor of the sample multiplied by ). If all dilutions are positive, the endotoxin
concentration is reported as equal to or greater than the largest dilution factor multiplied by λ
(e.g. initial dilution factor multiplied by 8 and by λ in Table 3).

The preparation under test meets the requirements of the test if the concentration of endotoxin
in both replicates is less than that specified in the individual monograph.

PHOTOMETRIC QUANTITATIVE TECHNIQUES

METHOD B. TURBIDIMETRIC TECHNIQUE

This technique is a photometric assay measuring increases in reactant turbidity. On the basis
of the particular assay principle employed, this technique may be classified as either an
endpoint-turbidimetric assay or a kinetic-turbidimetric assay.

The endpoint-turbidimetric assay is based on the quantitative relationship between the

concentration of endotoxins and the turbidity (absorbance or transmission) of the reaction
mixture at the end of an incubation period.

The kinetic-turbidimetric assay is a method to measure either the time (onset time) needed to
reach a predetermined absorbance or transmission of the reaction mixture, or the rate of
turbidity development.

The test is carried out at the incubation temperature recommended by the lysate manufacturer
(which is usually 37 ± 1°C).
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METHOD C. CHROMOGENIC TECHNIQUE

This technique is an assay to measure the chromophore released from a suitable chromogenic
peptide by the reaction of endotoxins with lysate.

On the basis of the particular assay principle employed, this technique may be classified as
either an endpoint-chromogenic assay or a kinetic-chromogenic assay.

The endpoint-chromogenic assay is based on the quantitative relationship between the

concentration of endotoxins and the release of chromophore at the end of an incubation period.

The kinetic-chromogenic assay is a method to measure either the time (onset time) needed to
reach a predetermined absorbance of the reaction mixture, or the rate of color development.

The test is carried out at the incubation temperature recommended by the lysate manufacturer
(which is usually 37 ± 1°C).

Preparatory testing

To assure the precision or validity of the turbidimetric and chromogenic techniques,

preparatory tests are conducted to show that the criteria for the standard curve are valid and
that the sample solution does not interfere with the test.

Validation for the test method is required when conditions change which are likely to influence
the result of the test.

Assurance of criteria for the standard curve

The test must be carried out for each lot of the lysate. Using the Standard Endotoxin Solution,
prepare at least three endotoxin concentrations within the range indicated by the lysate
manufacturer to generate the standard curve. Perform the assay using at least three replicates
of each standard endotoxin concentration according to the manufacturer's instructions for the
lysate (volume ratios, incubation time, temperature, pH etc.).
If the desired range is greater than two logs in the kinetic methods, additional standards should
be included to bracket each log increase in the range of the standard curve.

The absolute value of the correlation coefficient, |r|, must be greater than or equal to 0.980, for
the range of endotoxin concentrations set up.

Test for interfering factors

Select an endotoxin concentration at or near the middle of the endotoxin standard curve.
Prepare solutions A, B, C and D shown in Table 4. Perform the test on solutions A-D in at
least duplicate according to the instructions for the lysate employed, for example, concerning
volume of sample solution and lysate TS, volume ratio of sample solution to lysate TS,
incubation time, etc.
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Table 4
Solution Endotoxin concentration Solution to which endotoxin Number of
is added replicates

A None Sample solution Not less than 2

B Middle concentration of the Not less than 2

Sample solution
standard curve

At least 3 concentrations (lowest Each not less

C Water BET
concentration is designated λ) than 2

D None Water BET Not less than 2

Note:
- Solution A : The sample solution may be diluted not to exceed the MVD.
- Solution B : The preparation under test at the same dilution as solution A, containing
added endotoxin at a concentration equal to or near the middle of the standard curve.
- Solution C : The standard endotoxin at the concentrations used in the validation of the
method described in Assurance of criteria for the standard curve under Preparatory
testing (positive controls)
- Solution D : Water BET (negative control)

The test is considered valid when the following conditions are met.

1: The absolute value of the correlation coefficient of the standard curve generated using
solution C is greater than or equal to 0.980.
2: The result with solution D does not exceed the limit of the blank value required in the
description of the lysate employed, or it is less than the endotoxin detection limit of the
lysate employed.

Calculate the mean recovery of the added endotoxin by subtracting the mean endotoxin
concentration in the solution, if any (Solution A, Table 4), from that containing the added
endotoxin (Solution B, Table 4).

In order to be considered free of factors that interfere with the assay under the conditions of the
test, the measured concentration of the endotoxin added to the sample solution must be within
50-200% of the known added endotoxin concentration after subtraction of any endotoxin
detected in the solution without added endotoxin.

When the endotoxin recovery is out of the specified range, the sample solution under test is
considered to contain interfering factors. Then repeat the test using a greater dilution, not
exceeding the MVD. Furthermore, interference of the sample solution or diluted sample
solution not to exceed the MVD may be eliminated by suitable validated treatment, such as
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filtration, neutralization, dialysis or heat treatment. To establish that the treatment chosen
effectively eliminates interference without loss of endotoxins, perform the assay described
above using the preparation to be examined to which standard endotoxin has been added and
which has then been submitted to the chosen treatment.

Test

Procedure
Follow the procedure described in Test for interfering factors under Preparatory testing.

Calculation
Calculate the endotoxin concentration of each of the replicates of test solution A using the
standard curve generated by the positive control solution C. The test is considered valid when
the following three requirements are met.

1: The results of the positive control solution C comply with the requirements for validation
defined in Assurance of criteria for the standard curve under Preparatory testing.

2: The endotoxin recovery, calculated from the concentration found in solution B after
subtracting the concentration of endotoxin found in the solution A, is within the range of
50-200%.

3: The result of the negative control solution D does not exceed the limit of the blank value
required in the description of the lysate employed, or it is less than the endotoxin detection
limit of the lysate employed.

Interpretation
In photometric assays, the preparation under test complies with the test if the mean endotoxin
concentration of the replicates of solution A, after correction for dilution and concentration, is
less than the endotoxin limit for the product.

***

New/revised reagents to be added to PhInt

Amoebocyte lysate
A lyophilized product obtained from the lysate of amebocytes (white blood cells) from the
horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).
Note: Amebocyte lysate reacts to some β-glucans in addition to endotoxins. Amebocyte lysate
preparations which do not react to glucans are available: they are prepared by removing the G
factor reacting to glucans from amebocyte lysate or by inhibiting the G factor reacting system
of amebocyte lysate and may be used for the endotoxin testing in the presence of glucans.
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Lysate TS
Dissolve amebocyte lysate in water BET or in a buffer recommended by the lysate
manufacturer, by gentle stirring. Store the reconstituted lysate, refrigerated or frozen, according
to the specifications of the manufacturer.

Water BET
Water for injections or water produced by other procedures that shows no reaction with the
lysate employed, at the detection limit of the reagent.

***